CN105543249B - A kind of beauty Storehouse midge specific gene and its method for identifying molecules - Google Patents
A kind of beauty Storehouse midge specific gene and its method for identifying molecules Download PDFInfo
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- CN105543249B CN105543249B CN201610011293.2A CN201610011293A CN105543249B CN 105543249 B CN105543249 B CN 105543249B CN 201610011293 A CN201610011293 A CN 201610011293A CN 105543249 B CN105543249 B CN 105543249B
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Abstract
The present invention discloses a kind of beautiful Storehouse midge specific gene and its method for identifying molecules, it is characterised in that I gene order of CO that the Storehouse midge is obtained using molecular biology method carries out Identification of Species to the Storehouse midge by the comparison of gene order similitude.Beauty Storehouse midge method for identifying molecules provided by the invention, is advantageously implemented beautiful Storehouse midge and accurately and rapidly identifies.
Description
Technical field
The present invention relates to species identification fields, and in particular to a kind of I gene order of CO and its method for identifying molecules of Storehouse midge.
Background technique
Storehouse midge is one of big blood sucking midge in China three, can propagate blue tongue disease, live in a variety of Amphixenosis such as globidiosis, is a kind of
Important Vector.Therefore, to the identification of blood sucking midge and identification, it is that monitoring with control midge passes the pathogenetic basis of disease, and
The core procedure in prevention and control of diseases field.Know currently, relying primarily on veteran taxology expert to the identification of blood sucking midge
Other morphological feature realizes, once encounter sibling species and morphological feature grasp it is very intractable not full Specimen identification；Moreover, right
The Morphological Identification of blood sucking midge needs to complete by production slide sample, complex for operation step, the period is long, difficulty is big, therefore, urgently
It need to seek a kind of new method, to make up the defect of conventional sorting methods.
DNA bar code (DNA barcode), which refers to, can represent the species in organism, standard, have enough variations
, easily amplification and relatively short DNA fragmentation.DNA bar code technology is using one section of conservative fragments in organism DNA to species
The emerging technology quick and precisely identified.In recent years, DNA bar code technology had proved to be an effective biology
Identification of means can not only make strong supplement to conventional identification method, but also because it is more objective, accurate, it is previous right to break through
Experience is depended on unduly, be conducive to help identify species, discovery novel species and hidden kind, rebuild species and higher level taxa Evolvement
Deng.
It as the standard target gene of DNA bar code, on the one hand must guard enough, convenient for being carried out using universal primer
Amplification on a large scale；On the other hand there are enough variations to distinguish different plant species.Therefore, different species monoid needs are identified
Select effective target gene.Currently, widely applied marker gene is ribosomes 12S and 16S base in systematic growth research
Cause, but because it has a large amount of insertion and deficient phenomena, bar coding gene should not be used as.13 present in mitochondrial genomes
Only cytochrome oxidase subunit I (Cytochrome Oxidase Subunit I, CO I), line in a protein coding gene
Mitochondrial cytochrome b(Cytb), ND4, ND5 this 4 genes meet gene insertion and deficient phenomena is less, length is 900
The two conditions of bp or so, but ND4 and ND5 evolution is too fast, it is impossible to universal primer is designed, it is limited
Target gene as comprehensive DNA identification systems.Old friends finally select a segment in I gene of CO as DNA item
Shape encoding gene.So far, having many researchs has shown that I gene of CO in birds, fish, lepidopterous insects, mosquito class, ant class
With it is effective in the taxonomic identification of the monoids such as Collembola.Therefore, the DNA bar codes technique pair based on I gene of CO can be used
Blood sucking midge carries out Molecular Identification.This method is proved each other with traditional classification method, can not only solve identification in sample incompleteness not
Congruent problem, and can realize the Rapid identification of blood sucking midge.Currently, having obtained I gene of CO of a variety of blood sucking midges by this method
Sequence simultaneously uploads in Genbank, but there is no the related gene sequence of beautiful Storehouse midge so far.Beauty Storehouse midge gene provided by the invention
Sequence is advantageously implemented the Rapid identification of beautiful Storehouse midge, shortens qualification time.
Summary of the invention
The purpose of the present invention is to provide a kind of I gene orders of CO of Storehouse midge.It is obtained by molecular biology method beautiful
Storehouse midge (Culicoides bellulus) I gene order of CO can be accurately and rapidly by the comparison of gene order similitude
Identify beautiful Storehouse midge.
To achieve the above object, the present invention is achieved through the following technical solutions:
Using small insects minim DNA method for preparing template, by improved PCR reaction condition, by the primer synthesized
I gene of CO of the Storehouse midge is expanded, PCR product sequence is sent to be measured by professional biotech firm.Sequencing result passes through check and correction, sequence by hand
Splicing, and Blast similarity searching is carried out in NCBI, it is ensured that gained sequence is target sequence.Beauty of the present invention
Storehouse midge specific gene is I gene of CO, and I gene order of CO of the Storehouse midge (is free of primer) as shown in SEQ ID No.1:
The PCR primer of I gene of beauty Storehouse midge CO of the present invention, it is characterised in that PCR primer sequence are as follows:
5 ' AATCATAAAGATATTGGAAC 3 ' of forward primer
5 ' CCAAAAAATCAAAATAAATGTTG 3 ' of reverse primer.
The method for identifying molecules of beauty Storehouse midge of the present invention, comprising:
1) the separation and Extraction DNA from insect tissue to be measured；
2) using the DNA as template, the primer of use are as follows: 5 ' AATCATAAAGATATTGGAAC 3 ' of forward primer, reversely
5 ' CCAAAAAATCAAAATAAATGTTG 3 ' of primer goes out I gene of CO of the insect by polymerase chain reaction amplification；
3) I gene of CO for then taking the polymerase chain reaction amplification of appropriate step 2 to go out is separated with agarose electrophoresis, is passed through
It is observed under ultraviolet lamp after ethidium bromide staining, result is determined according to the size of amplified production.If can correspondingly specific amplification go out
Size is about the band of 658 bp, and biotech firm is sent to be sequenced；
4) according to sequencing result, if the similitude of target gene sequence and SEQ ID No.1 98% or more, that is, can determine whether
The test serum is beautiful Storehouse midge.
The Identification of Species of beautiful Storehouse midge is realized by Morphological Identification, and is checked through authoritative expert, to guarantee to finish
The reliability of fruit.
The primer is synthesized according to document, 5 ' AATCATAAAGATATTGGAAC 3 ' of forward primer, reverse primer 5 '
CCAAAAAATCAAAATAAATGTTG 3’。
The present invention can detect amplification with agarose gel electrophoresis detection method.
Advantages of the present invention are as follows:
1) present invention is combined using traditional Morphological Identification with molecular classification method, is identified the Storehouse midge,
It can ensure that the accuracy and reliability of species identification.
2) compared with traditional Morphological Identification method, the beautiful Storehouse midge method for identifying molecules that the present invention establishes can effectively contract
The qualification time of short beauty Storehouse midge.
Detailed description of the invention
Fig. 1 is beautiful I gene PCR amplification electrophoretic identification of Storehouse midge CO.Number is described as follows: swimming lane 1 is negative right
According to 2-5 is I gene of CO of beautiful Storehouse midge female adult, the band that detection size is about 658 bp.M is the DNA of DL 2000
marker。
Fig. 2 is unknown I gene PCR amplification electrophoretic identification of Storehouse midge female adult CO.Number is described as follows: swimming lane 1 is yin
Property control, 2 be unknown Storehouse midge female adult I gene of CO, detection size be about 658 bp band.M is the DNA of DL 2000
marker。
Specific embodiment
The beautiful Storehouse midge of embodiment 1 (Culicoides bellulus) CO I gene order acquisition
1, the acquisition of beautiful Storehouse midge sample
Beautiful Storehouse midge is the sample that field acquisition obtains, and is directly stored in 95% alcohol after chilled execution.Sample is made
It is identified after slide through authoritative expert, it is ensured that the accuracy of qualification result.Before preparation of specimen, picking beauty Storehouse midge chest end and abdomen
The former sections in portion are placed in 95% alcohol, are extracted for DNA.
2, prepared by DNA profiling
Beauty Storehouse midge DNA(Wen Lizhang is extracted using small insects trace DNA templet preparation method and edits entomology research
Method and the Beijing technology introduction [M]: Science Press, 2010.278-286), the DNA sample of extraction is standby in -20 DEG C of preservations
With.
3, primer synthesizes
The present embodiment the primer is as follows:
5 ' AATCATAAAGATATTGGAAC 3 ' of forward primer
5 ' CCAAAAAATCAAAATAAATGTTG 3 ' of reverse primer.
4, PCR amplification
The PCR reaction system of the present embodiment is as shown in table 1.
The beautiful I gene PCR reaction system (50uL system) of Storehouse midge CO of table 1
The response procedures of the present embodiment are as shown in table 2.
The beautiful I gene PCR response procedures of Storehouse midge CO of table 2
PCR product is saved backup in 4 DEG C.
5, pcr amplification product detects
5 μ L pcr amplification products are taken, with 1% agarose gel electrophoresis (130V voltage, 35 min of electrophoresis).Ethidium bromide staining,
Gel imaging system detection, electrophorogram is referring to attached drawing 1.
6, gene sequencing
Choose the preferable pcr amplification product of 3-5 effect send and be sequenced after purification by biotech firm (be sequenced the primer and
PCR the primer is identical), it is the CO of beautiful Storehouse midge after the calibrated splicing of gained gene orderGene order-SEQ ID
No.1。
The identification of the unknown Storehouse midge of embodiment 2
1, the acquisition and preservation of sample
Using net method is waved or lamp lures method to be acquired, directly it is stored in 95% alcohol after the chilled execution of the sample of harvest.
2, prepared by DNA profiling
Under anatomical lens or stereomicroscope, picks out a female Storehouse midge at random from collected midge class sample, adopt
Storehouse midge DNA(Wen Lizhang chief editor's entomology research method and technology introduction are extracted with small insects trace DNA templet preparation method
Beijing [M]: Science Press, 2010.278-286), the DNA sample of extraction is saved backup in -20 DEG C, and every part sample one
Number.
3, the acquisition of objective gene sequence
The synthesis of 3.1 primers
Using the unknown Storehouse midge DNA of acquisition as template, synthetic primer, the primer sequence is as follows:
5 ' AATCATAAAGATATTGGAAC 3 ' of forward primer
5 ' CCAAAAAATCAAAATAAATGTTG 3 ' of reverse primer.
3.2 PCR amplification
The PCR reaction system of the present embodiment is as shown in table 3.
The unknown I gene PCR reaction system of Storehouse midge CO (50uL system) of table 3
| Ingredient | Volume (uL) |
| Template | 2 |
| Primer 1 | 1 |
| Primer 2 | 1 |
| 10 × buffer | 5 |
| dNTPs | 1 |
| Taq archaeal dna polymerase | 2.5 |
| ddH2O | 37.5 |
The response procedures of the present embodiment are as shown in table 4.
The unknown I gene PCR response procedures of Storehouse midge CO of table 4
| Step | Reaction temperature (DEG C) | Time | Recurring number |
| Initial denaturation | 94 | 3 min | — |
| Denaturation | 94 | 45 s | 35 |
| Annealing | 48 | 45 s | 35 |
| Extend | 72 | 60 s | 35 |
| Extend | 72 | 7 min | — |
PCR product is saved backup in 4 DEG C.
The detection of 3.3 pcr amplification products and gene sequencing
5 μ L pcr amplification products are taken, with 1% agarose gel electrophoresis (130V voltage, 35 min of electrophoresis).Ethidium bromide dye
Color.Gel imaging system detection, electrophorogram is referring to attached drawing 2.Find out that the unknown Storehouse midge of embodiment 2 also can specificity by attached drawing 2
Amplify the product that size is about 658 bp, send biotech firm to be sequenced the product that size is about 658 bp, as the result is shown with
The similitude of gene order SEQ ID NO. 1 is 99.9%, thus can determine that the unknown Storehouse midge for beautiful Storehouse midge.
Claims (1)
1. a kind of beauty Storehouse midge specific gene, which is characterized in that the special gene sequence is I gene of CO, is as described below
Gene order SEQ ID No.1:
。
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988232A (en) * | 2018-01-25 | 2018-05-04 | 遵义医学院 | The DNA bar code standard sequence and its method for identifying molecules of a kind of open country Storehouse midge |
| CN108486121B (en) * | 2018-01-25 | 2020-02-11 | 遵义医学院 | Specific DNA sequence of Cuicoides spinuloides and molecular identification method thereof |
| CN108034733B (en) * | 2018-01-29 | 2020-05-05 | 遵义医学院 | DNA bar code standard sequence of Culicoides kindred species and molecular identification method thereof |
| CN108486122A (en) * | 2018-01-29 | 2018-09-04 | 遵义医学院 | A kind of the DNA bar code standard sequence and its method for identifying molecules of South Mountain Storehouse midge |
| CN108342398A (en) * | 2018-02-08 | 2018-07-31 | 南昌市疾病预防控制中心 | A kind of specific gene and its method for identifying molecules of open country Storehouse midge |
| CN109706251B (en) * | 2019-03-13 | 2022-03-11 | 河北国际旅行卫生保健中心 | Kit and method for identifying culicoides flavipes |
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| CN103805609A (en) * | 2014-03-03 | 2014-05-21 | 福建国际旅行卫生保健中心 | Molecular identification method for lasiohelea taiwana |
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| CN103805609A (en) * | 2014-03-03 | 2014-05-21 | 福建国际旅行卫生保健中心 | Molecular identification method for lasiohelea taiwana |
Non-Patent Citations (2)
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| "DNA条形码技术应用于口岸吸血蠓类快速分子鉴定的研究";贺骥 等;《中华卫生杀虫药械》;20140430;第20卷(第2期);第131-135页 |
| "Taxonomy and biology of a new ambrosia gall midge Daphnephila urnicola sp.nov. (Diptera: Cecidomyiidae) inducing urn-shaped leaf galls on two species of Machilus (Lauraceae) in Taiwan";LIANG-YU PAN 等;《Zootaxa》;20150505;第3955卷(第3期);第374页第一段 |
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